Whole genome amplification of the obligate intracellular pathogen Coxiella burnetii using multiple displacement amplification

Kumar, Sanjay; Gangoliya, Shefali Raj; Berri, Mustapha; Rodolakis, Annie; Alam, Syed Imteyaz

doi:10.1016/j.mimet.2013.10.008

Cited by 3 publications

(2 citation statements)

References 15 publications

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“…MDA with random hexamers has been used to amplify Xylella fastidiosa (Gram negative plant pathogen, 52% CG content) DNA directly from approximately 1000 target cells, yielding over 4 μg of high molecular weight DNA and achieving uniform genome coverage relative to unamplified DNA [34]. Coverage of Coxiella burnetii (fastidious obligate intracellular pathogen, 42.5% GC) was similarly representative, as assessed by PCR [36].…”

Section: Discussionmentioning

confidence: 99%

MinION Nanopore Sequencing of Multiple Displacement Amplified Mycobacteria DNA Direct from Sputum

George

Sanderson

et al. 2018

Preprint

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37Sequencing of pathogen DNA directly from clinical samples offers the possibilities of rapid 38 diagnosis, faster antimicrobial resistance prediction and enhanced outbreak investigation. The 39 approach is especially advantageous for infections caused by species which grow very slowly 40 in culture, such as Mycobacteria tuberculosis. Since the pathogen of interest may represent as 41 little as 0.01% of the total DNA, enrichment of the input material for target sequences by 42 specific amplification and, or depletion of non-target DNA (human, other bacteria) is 43 essential for success. Here, we investigated the potential of isothermal multiple displacement 44 amplification by Phi29 polymerase. We directed the amplification reaction towards 45 Mycobacteria DNA in sputum samples by exploiting in our oligonucleotide primer design, 46 their high GC content (approximately 65%) relative to human DNA. Amplified DNA was 47 then sequenced using the Oxford Nanopore Technology MinION. In addition, a model 48 system comprising standardised 'mock clinical samples' was designed. Pooled infection 49 negative human sputum samples were spiked with enumerated Mycobacterium bovis (BCG) 50 Pasteur strain at concentrations spanning the typical range at which Mycobacterium 51 tuberculosis is found in human sputum samples (10 6 -10 1 BCG cells/ml). To assess the 52 amount of BCG sequence enrichment achieved, sample DNA was sequenced both before, and 53 after amplification. Reads from amplified samples, which mapped to a BCG reference 54 genome, comprised short repeated sequences -apparently transcribed multiple times from the 55 same fragment of BCG DNA. Therefore post-amplification, the samples were enriched for 56 BCG sequences relative to unamplified sequences (8,101 BCG reference mapped reads, 57 increasing to 28,617 at 10 6 BCG cells/ml sample), but BCG genome coverage declined 58 markedly (for example 89.4% to 4.1%). In summary, the use of standardised mock clinical 59 samples allowed direct comparison of data from different Mycobacteria enrichment 3 60 experiments and sequencing runs. However, optimal conditions for multiple displacement 61 amplification of minority Mycobacteria DNAs, remain to be identified. 4 62 5 87 concentration of target organisms. Mycobacteria DNA, for example, can represent as little as 88 0.01% of the total DNA extracted from sputum [11]. Small scale studies employing direct 89 from sample sequencing have reported 0.002 -0.7% sequence coverage of the M. 90 tuberculosis genome (using differential lysis and a DNA extraction kit) [12], and up to 90% 91 genome coverage with 20x depth (20/24 samples), (using the SureSelect target enrichment 92 method, Agilent, USA) [13, 14]. The study by Brown et al. [13] predicted both Mycobacteria 93 species and antibiotic susceptibility, but the cost ($350 per sample) and duration of the 94 protocol (2 to 3 days) could prevent its use. The ideal 'direct from sample' methodology 95 would be simple, low cost and portable, to facilitate use in remote, low-income settings 96 ...

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Section: Discussionmentioning

confidence: 99%

MinION Nanopore Sequencing of Multiple Displacement Amplified Mycobacteria DNA Direct from Sputum

George

Sanderson

et al. 2018

Preprint

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“…We chose a special variant of WGA, the MDA method, that has been successful applied [ 3 , 4 ] and is commercially available from different companies (RepliG, Qiagen, Hilden, Germany and GenomiPhi, GE Healthcare, Freiburg, Germany) [ 5 ]. Very recently the RepliG kit was used with Coxiella DNA and evaluated at 20 selected loci [ 6 ].…”

Section: Introductionmentioning

confidence: 99%

Genome sequence of Coxiella burnetii strain Namibia

Walter

Öhrman

Myrtennäs

et al. 2014

Stand in Genomic Sci

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We present the whole genome sequence and annotation of the Coxiella burnetii strain Namibia. This strain was isolated from an aborting goat in 1991 in Windhoek, Namibia. The plasmid type QpRS was confirmed in our work. Further genomic typing placed the strain into a unique genomic group. The genome sequence is 2,101,438 bp long and contains 1,979 protein-coding and 51 RNA genes, including one rRNA operon. To overcome the poor yield from cell culture systems, an additional DNA enrichment with whole genome amplification (WGA) methods was applied. We describe a bioinformatics pipeline for improved genome assembly including several filters with a special focus on WGA characteristics.

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